Stem cell properties in cell cultures from different stage of melanoma progression.

Cutaneous melanoma is an extremely heterogenous human cancer. The most aggressive melanoma may contain deregulated cells with undifferentiated/stem cell-like phenotype. A critical mechanism by which melanoma cells enhance their invasive capacity is the dissolution of the intercellular adhesion and the acquisition of mesenchymal features as a part of an epithelial-to-mesenchymal transition. The aim of this study was to clarify the role of a stem cell-like population in human melanomas by means of melanocytic cell culture analysis obtained from distinct histotypes of primary and metastatic malignant melanoma. Patients with advanced melanoma >2 cm in diameter and/or >300 mm surface were enrolled. The melanoma cells were isolated from skin biopsies of lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, and metastatic melanoma. The colony forming unit assay and alkaline phosphatase stain were evaluated. Cells were subsequently cultured and maintained in different media to evaluate their ability to differentiate into osteogenic and adipogenic lineages. Immunohistochemistry and flow cytometry analysis were performed to evaluate antigenic markers CD90, CD73, CD105, CD146, CD20, CD166, and Nestin. This study confirms that melanoma can include heterogenous cell populations with the ability both to self-renew and to a give rise to differentiated progeny. Melanoma cells displayed intratumoral heterogeneity and dynamic antigen phenotypes. Histologically, transitions from normal skin to melanoma were associated with a gradual increase in the expression of CD146, CD20, CD133, Nestin, and CD73. These molecular profiles could be further analyzed and, in the future, used for the development of novel biomolecular targeted-therapy approaches.

Biological evaluation of MR36, a novel non-polyglutamatable thymidylate synthase inhibitor that blocks cell cycle progression in melanoma cell lines.

Melanoma is one of the most common cancers, and its incidence has continued to increase over the past few decades. Chemotherapy resistance and related defects in apoptotic signaling are critical for the high mortality of melanoma. Effective drugs are lacking because apoptosis regulation in this tumor type is not well understood. The folate pathway has been considered an interesting target for anticancer therapies, and approaches targeting this pathway have recently been extended to melanoma treatment. In this study, the intracellular apoptosis signaling pathways of two melanoma cells lines (SK-MEL-2 and SK-MEL-28) were investigated after treatment with a new experimental antifolate substance (MR36) that targets thymidylate synthase. In both melanoma cell lines, apoptosis induction was triggered by a p53-independent mechanism. MR36-induced apoptosis was associated with a loss of both mitochondrial membrane potential and caspase-3 activation. Induction of cell cycle arrest by MR36 was associated with changes in the expression of key cell cycle regulators, such as p21 and cyclin D1, and the hypophosphorylation of pRb. In addition, Fas signaling was also analyzed. These findings suggest that, unlike classical antifolates, MR36 exerted an inhibitory effect on both the enzymatic function and expression of thymidylate synthase, thereby inducing apoptosis through the activation of the extrinsic and intrinsic pathways in the melanoma cell lines. MR36 showed a different mechanism of action from the known antifolates (Nolatrexed and Pemetrexed) that resulted in higher anticancer activity. Therefore, MR36 should be included as a potential new therapeutic treatment in melanoma research.

Brooke-Spiegler syndrome: report of two cases not associated with a mutation in the CYLD and PTCH tumor-suppressor genes.

Brooke-Spiegler syndrome represents an autosomal dominant disease characterized by the occurrence of multiple cylindromas, trichoepitheliomas and (sporadically) spiroadenomas. Patients with Brooke-Spiegler syndrome are also at risk of developing tumors of the major and minor salivary glands. Patients with Brooke-Spiegler syndrome have various mutations in the CYLD gene, a tumor-suppressor gene located on chromosome 16q. To date, 68 unique CYLD mutations have been identified. We describe two families with Brooke-Spiegler syndrome, one with familial cylindromatosis and one with multiple familial trichoepithelioma, which showed wide inter-family phenotypic variability. Analysis of germline mutations of the CYLD and PTCH genes was performed using peripheral blood. In addition, formalin-fixed paraffin-embedded tumor samples were analyzed for PTCH somatic mutations and cylindroma cell cultures were obtained directly from patients for further growth and analysis. Clinically, the major features of Brooke-Spiegler syndrome include the presence of heterogeneous skin tumors and wide inter- and intra-familial phenotypic variability. Histopathologically, both cylindromas and trichoepitheliomas were found in affected individuals. Mutations or loss of heterozygosity was not found in CYLD and PTCH genes. In CYLD and PTCH mutation-negative patients, other genes may be affected and further studies are needed to clarify whether these patients may be affected by de novo germline mutations.

18beta-glycyrrhetinic acid and glabridin prevent oxidative DNA fragmentation in UVB-irradiated human keratinocyte cultures.

BACKGROUND: UVB radiation is the major etiological factor in the pathogenesis of skin aging and cancer development. New approaches to prevent and reverse UVB damage are needed to reduce sunlight-induced skin cancer. This study aimed to investigate a possible protective activity of liquorice root extracts glycyrrhizin (GL), 18ß-glycyrrhetinic acid (18ß-GA) and glabridin (GLB) against UVB radiation damage in human keratinocyte cultures. MATERIALS AND METHODS: The MTT test was performed to assess cell viability. DNA damage was evaluated by comet assay, whereas generation of intracellular reactive oxygen species (ROS) was measured by fluorescent 2'7'-dichlorodihydrofluorescein diacetate assay. In addition, the activation of p53, regulation of BCL-2 and PARP cleavage were analyzed by Western blot analysis. RESULTS: The treatment of human keratinocytes with 18ß-GA and GLB prevented direct and indirect DNA damage avoiding apoptosis activation. CONCLUSION: 18ß-glycyrrhetinic acid and glabridin are potent antioxidants that prevent oxidative DNA fragmentation and the activation of apoptosis-associated proteins in human keratinocytes.

Mepacrine antagonises tumour cell growth induced by natural polyamines.

BACKGROUND: Mepacrine is an antiproliferative agent, characterised by an aliphatic chain similar to that of natural polyamines whose activation is closely associated with cell proliferation and may lead to malignant transformation and neurodegenerative diseases. This study aims to investigate a possible antagonism between mepacrine and polyamines in tumour proliferation. MATERIALS AND METHODS: MCF-7 and Vero cells were cultured in Eagle's minimum essential medium and then subjected to graded concentrations of putrescine, spermine and spermidine alone and in combination with mepacrine. Methyl thiazole tetrazolium test and Western-blotting were performed. RESULTS: Putrescine and spermidine at 0.5 mg/l significantly stimulated cell growth, whereas mepacrine treatment confirmed the enhanced p21 expression previously reported by a recent study and growth inhibition. When used in combination, mepacrine antagonized MCF-7 growth induced by polyamines. CONCLUSION: Our results suggest that mepacrine may represent a choice in the treatment of tumours induced by the modified concentration of polyamines.

Pharmacological and toxicological evaluation of a new series of thymidylate synthase inhibitors as anticancer agents.

Thymidylate synthase (TS) is responsible for catalysing the de novo biosynthesis of doexythymidine monophosphate and is a target for many anticancer drugs. A series of thymidylate synthase inhibitors (TSIs), synthesised in our laboratory, were submitted to primary anticancer screening by the National Cancer Institute (NCI). Four compounds, 3,3-bis(4-methoxyphenyl)-1H, 3H-naphtho[1,8-cd]pyran-1-one (MR7), 6-chloro-3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR21), 3,3-bis(3-fluoro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR35) and 6-bromo-3,3-bis(3-chloro-4-hydroxyphenyl)-1H,3H-naphtho[1,8-cd]pyran-1-one (MR36), passed the criteria and were automatically scheduled for evaluation against the full panel of 60 human tumour cell lines. In this study, the antiproliferative activity of the substances against SK-MEL-2 cells (from metastatic tissue) and SK-MEL-28 cells (from primary malignant melanoma cells) was investigated. Neutral Red uptake and the MTT test were performed to confirm the results of the NCI, and [3H]-thymidine incorporation was performed as a test of the proliferation rate. Our results indicated that compounds MR21 and MR36 were the most active agents and the [3H]-thymidine test was the best in predicting toxicity against melanoma cells.